Heteromultimeric ion channel receptor and uses thereof

ABSTRACT

The present invention describes a heteromultimeric proton-gated ion channel (herein called ASIC-2S.2) with distinctive properties. Compositions and methods are provided for producing and expressing functional ASIC-2S.2 channels, composed of ASIC2A and ASIC3 subunits. The invention also provides genetically engineered expression vectors comprising the nucleic acid sequences encoding both ASIC2A and ASIC3 and host cells coexpressing both ASIC2A and ASIC3 subunits. Also provided herein are genetically engineered nucleic acids encoding chimeric proton-gated ion channels comprised of at least two different subunits, as well as expression vectors and host cells comprising said engineered nucleic acids. The invention also provides for the use of ASIC-2S.2, as well as agonists, antagonists or antibodies specifically binding ASIC-2S.2, in the diagnosis, prevention and treatment of diseases associated with expression of ASIC-2S.2. Also are disclosed methods of influencing electrophysiological, pharmacological and/or functional properties of ASIC-2S.2 as well as methods for screening for substances having ion-channel modulating activity or substances capable of disrupting subunit association or interaction.

FIELD OF THE INVENTION

The present invention is based on the discovery of a novel Acid SensingIon Channel (ASIC) with distinctive channel properties and biologicalactivity. This novel channel, ASIC-2S.2 is a heteromultimeric complexcomprised of two different types of ASIC subunits, namely ASIC2A andASIC3. The present invention is also based on the discovery of a new useof polynucleotides encoding ASIC2A and ASIC3, said use being theinclusion of ASIC2A and ASIC3 in the assembly of a heteromultimeric ionchannel. This invention further includes the use of the abovecompositions for diagnosis, prevention, or treatment of diseases relatedto the expression of the heteromultimeric ASIC channel disclosed herein.

The present invention demonstrates for the first time the directbiochemical interaction between two distinct ASIC subunits, namelyASIC2A and ASIC3, which produces a novel proton-gated ion channel withdistinctive properties.

BACKGROUND OF INVENTION

In mammals, the pH of the extracellular compartment, includinginterstitial fluids and blood, is strictly regulated and maintained at aconstant value of 7.4. Acid sensing is a specific kind of chemoreceptionthat plays a critical role in the detection of nociceptive pH imbalancesoccurring, for example, in conditions of cramps, trauma, inflammation orhypoxia (Lindahl, Adv Neurol 1974; 4: 45)). In mammals, a population ofsmall-diameter primary sensory neurons in the dorsal root ganglia andtrigeminal ganglia (Bevan and Yeats, J Physiol (Lond) 1991; 433: 145) aswell as central neurons (Varming, Neuropharmacol 1999; 38: 1875) expressspecialized pH-sensitive surface receptors activated by an increase ofextracellular proton concentrations. Acid sensitivity of sensory as wellas central neurons is mediated by a family of proton-gated cationchannels structurally related to C. elegans degenerins (DEG) andmammalian epithelial sodium channels (ENaC). This invention relates tothese Acid Sensing Ion Channels (ASIC) and specifically reports thediscovery of novel class of receptors generated by the heteromultimericassembly of two distinct ASIC subunits, namely ASIC2A (or BnaC1, orBNC1, or MDEG, or MDEG1) and ASIC3 (or hASIC3, or DRASIC) and usesthereof.

Tissue acidosis is associated with a number of painful, physiological(e.g. cramps) and pathological conditions (e.g. inflammation,intermittent claudication, myocardial infarction). Experimentally,similar painful events can be reproduced by infusing low pH solutionsinto skin or muscle. Furthermore, the prolonged intradermal infusion oflow pH solutions can mimic the characteristic hyperalgesia of chronicpain. To further characterize the effects of protons and their relationto pain, low pH solutions were applied to patch-clamped central andperipheral sensory neurons. Inward currents were induced when pH wasdropped to acidic values, providing evidence for the existence ofproton-activated ion channels. Several types of native currents wereobserved in sensory neurons from rat and human trigeminal and dorsalroot ganglia as well as central neurons: rapidly inactivating currents;non-inactivating currents; and biphasic currents displaying a rapidlyinactivating current followed by non-inactivating sustained current.Other differences regarding ion selectivities were also reported. Theseresults suggested the existence of a multigene family of proton-gatedion channels, implicated in neurotransmission and/or neuromodulation.

Cloned Proton-gated Ion Channels

The mammalian proton-gated cation channels have recently been cloned andnamed <<ASIC>> for Acid Sensing Ion Channels. Sequence analysisidentifies them as members of the DEG/ENaC superfamily of ion channels.The putative membrane topology of ASIC receptors predicts twotransmembrane spanning domains with both N- and C-termini in theintracellular compartment, as shown for the epithelial sodium channels.Four sub-classes of ASIC receptors have been identified:

-   1. ASIC1 ion channels display rapidly inactivating inward currents    (Waldmann et al., Nature 1997; 386:173)-   2. ASIC2 ion channels display slowly inactivating inward currents    (Brassilana et al., J Biol Chem 1997; 272: 28819).-   3. ASIC3 ion channels display biphasic inward currents with an    initial rapidly inactivating component, followed by a sustained    non-inactivating current (Waldmann et al., J Biol Chem 1997; 272:    20975; Babinski et al., J Neurochem 1999; 72: 51)-   4. ASIC4 ion channels displaying similar properties as those of    ASIC3 (Wood et al., WO9963081)

Other recently discovered ion channel subunits, BLINaC and INaC, appearto belong to the ASIC family but are not activated by protons and havenot yet been shown to interact with other ASIC subunits (Sakai et al., JPhysiol 1999; 519: 323, Schaefer et al., FEBS Lett 2000; In Press).

Families of ASIC Receptors Created by Alternative Splicing of mRNAs

A common feature of these ion channels is the existence of alternativesplice variants, which display important functional differences. Indeed,the replacement of the first 185 amino acids of ASIC1 (hereinafter namedASIC1A) by a distinct new sequence of 172 amino acids generates a newchannel, ASIC1B, which has similar current kinetics as ASIC1A but needslower pH values for activation (pH₅₀ of 6.2 and 4.5, respectively, forASIC1A and ASIC1B). Also, it appears that ASIC1B is specificallyexpressed in rat dorsal root ganglia. A similar situation is alsoobserved with rat ASIC2 (hereinafter named ASIC2A), where thereplacement of the first 185 amino acids by a distinct new sequence of236 amino acids generates another ASIC ion channel subunit, ASIC2B. Whenexpressed alone as a homomultimer in mammalian cells or Xenopus oocytes,ASIC2B does not appear to be activated by low pH solutions. ASIC3, whichhas been identified in human, also appears to exist in various forms.Indeed, DRASIC is an ASIC3-like channel identified in rat, whichdisplays 85% identity with the human ASIC3 sequence and has similarbiphasic current kinetics. However, important differences regardingtissue distribution, ion selectivities and pH₅₀ suggest that DRASICmight not be the human orthologue of ASIC3 but rather a differentsubtype. Furthermore, the existence of two 3′ splice variants of humanASIC3 (ASIC3B and 3C, sequences submitted to GenBank) have been reportedbut differences in function have yet to be documented. Alternativesplicing, therefore, appears like an important mechanism for increasingthe diversity of ASIC receptors, which most probably assume criticalroles in the nervous system, such as neurotransmission, nociception ormechanosensation (see below).

Families of ASIC Receptors Created by Heteromultemeric Associations

In general, functional ion channels are complex structures comprised ofseveral individual components, referred) to as subunits. The number ofsubunits depends on the type of ion channel and subunits can either beall identical (homomultimeric channels) or include a combination ofseveral different subtypes (heteromultimeric channels). For example,Epithelial sodium Channels (ENaC), which belong to the same gene familyas ASIC receptors, are comprised of at least three different subunits,namely αEnaC, βEnaC and γEnaC (Canessa et al., Nature 1994; 367: 463).Although cloned ASIC receptors have mostly been characterized in vitroin their homomultimeric form, the analogy with EnaCs raises thepossibilty that ASIC subunits might also associate in variouscombinations to generate novel channels with distinctive properties.Indeed, heteromultimeric ASIC channels might account for some of thenative proton-gated currents still not explained by any of thehomomultimeric ASICs cloned to date. Examples of such native currentsare the sustained non-desensitizing currents seen at pH 6 (Bevan andYeats, J Physiol 1991; 433: 145). Furthermore, the discovery of theproton-insensitive ASIC2B (or MDEG2) suggests that it may function as anaccessory subunit. Indeed, the first evidence for heteromultimeric ASICreceptors came from coexpression studies featuring rat ASIC2B eitherwith ASIC2A or with ASIC3. Channels created by ASIC2A and ASIC2B appearto be slightly more sensitive to pH, while inward currents carried byASIC2B+ASIC3 channels are apparently less sodium selective than thehomomultimeric ASIC3 currents (Lingueglia et al., J Biol Chem 1997: 272:29778). However, no biochemical evidence of interaction has beenreported to date for any ASIC subunits. Furthermore, other coexpressionexperiments with different subunits suggest that not all subunitcombinations yield novel functional channels. Thus, the composition andfunctional characteristics of heteromultimeric ASIC channels aretherefore unpredictable.

SUMMARY OF THE INVENTION

It is the purpose of the present invention to disclose and describe anovel heteromultimeric ASIC channel, herein called ASIC-2S.2, and usesthereof.

The present invention reports the discovery of a novel heteromultimericASIC receptor (hereinafter called ASIC-2S.2). Also contemplated withinthe scope of this invention is the potential involvement of this newreceptor in neurotransmission and/or nociception and/or mechanosensationand/or any other neurological and/or metabolic processes in normaland/or pathophysiological conditions. This invention seeks also to coverany uses of this new ion channel as a therapeutic target, including butnot limited to drug screening technologies (i.e. screening for channelantagonists, agonists, modulators and/or subunit association blockers),diagnostic marker, or gene therapies. Also within the scope of thepresent invention is the heteropolymerization of the ASIC-2S.2 channelwith one or more additional subunits of the ASIC family from anyspecies, including but not limited to ASIC1, ASIC1A, BNaC2, ASIC1B,ASIC2B, MDEG2, ASIC4, SPASIC or any variants thereof, as well asheteropolymerization of ASIC-2S.2 with any other members of theDegenerin and EnaC family from any species.

An object of this invention is therefore to provide the composition ofthe novel ASIC-2S.2 receptor and methods of producing and expressingfunctional ASIC-2S.2 ion channels.

Another object of the present invention is to provide methods forengineering nucleic acids specifically designed to encode a chimericASIC receptor comprised of a single polypeptide, where two or more ASICsubunits are covalently linked together, and expressed in tandem as asingle amino acid sequence.

Also included within the scope of this invention are methods designedfor screening and identifying substances, whether chemically synthetisedor isolated from natural sources, which have ion channel modulatingactivity. Typically this includes but is not limited to competitive andnon-competitive agonists and partial agonists as well as competitive andnon-competitive antagonists and partial antagonists, as well anysubstance capable of directly or indirectly disrupting, inhibiting orpreventing the complete or partial association of ASIC subunits, asdisclosed hereinafter, into functional, partially functional, ornon-functional channels.

The invention additionally features the specific use of nucleic acidsencoding the ASIC subunits comprising the ASIC-2S.2 heteromultimericchannel, namely ASIC2A (BNaC1, MDEG1) and ASIC3 (or DRASIC), as well aspolypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments,portions, antisense molecules, or any derivatives thereof, wherespecific use includes disruption, inhibition or prevention of ASICsubunit association or assembly into the ASIC-2S.2 heteromultimericchannels of the present invention. Also within the scope of thisinvention are expression vectors and host cells comprising nucleic acidsthat simultaneously encode and/or express ASIC2A and ASIC3 subunitstogether. The present invention also features pharmaceuticalcompositions comprising substantially purified ASIC-2S.2 as well asantibodies which bind specifically to the ASIC subunits of the ASIC-2S.2channel complex and whose specific binding causes the disruption,inhibition or prevention of ASIC subunit association or assembly intothe ASIC-2S.2 heteromultimeric channels of the present invention

DESCRIPTION OF THE FIGURES

The following drawings, figures and tables are illustrative of theembodiments of the invention and are not meant to limit the scope of theinvention as encompassed by the claims.

FIG. 1 illustrates the pH-activated inward currents recorded in voltageclamped Xenopus oocytes expressing ASIC2A and ASIC3, either alone or incombination. The functional interaction is clearly visible whencomparing currents between mono-injected and co-injected oocytes. FIG.1A: Human subunits, BNaC1 and hASIC3; FIG. 1B: Rat subunits, MDEG1 andDRASIC.

FIG. 2 shows the pH dose-response curves of proton-induced inwardcurrents in human and rat homomultimeric (ASIC2A or ASIC3) andheteromultimeric (ASIC-2S.2)-expressing oocytes.

FIG. 3 shows the antagonistic effects of amiloride (A) and gadoliniumions (B) on proton-induced inward currents in human and rathomomultimeric (ASIC2A or ASIC3) and heteromultimeric(ASIC-2S.2)-expressing oocytes.

FIG. 4 represents an ethidium bromide stained agarose gel showing theexpression pattern of mRNA for hASIC2A and hASIC3 determined by duplexRT-PCR amplification of commercially available human RT-cDNA (Clontech)using specific oligonucleotide primers for each subunit. Noteworthy isthe co-expression of both subunits in trigeminal ganglia, suggestingthat the heteromultimeric receptor, ASIC-2S.2 might be involved in painand/or sensory transmission.

FIG. 5 reveals In situ hybridization in rat cerebellum with MDEG1- andDRASIC-specific probes showing the co-expression of both subunits in thesame cell type.

FIG. 6 illustrates the effects of N- or C-terminal tagging of ASIC2Aand/or ASIC3 subunits on proton-activated inward currents recorded usingvoltage clamped Xenopus oocytes expressing either the homomultimeric orheteromultimeric ASIC channels. FIG. 6A gives examples of currents andFIG. 6B summarises results in a table format.

FIG. 7 is a Western blot of N-terminally tagged hASIC2A and hASIC3subunits coexpressed in Xenopus oocytes (A) or HEK293 cells (B), showingthat both subunits are co-immunoprecipitated or co-purified, providingevidence in favour of a direct biochemical interaction between subunits.

FIG. 8 is a Table listing all the synonyms of ASIC2A and ASIC3 used invarious publications, databases, patent applications and patents.

FIG. 9 shows the inhibitory effects of C-terminal and N-terminalfragments of ASIC2A or ASIC3 on the current mediated by ASIC2S.2heteromoeric channel. Any given vectors carrying the fragments wasco-injected with the ASIC2A and ASIC3 subunits and tested at twodifferent ratios of fragment versus ASIC2A and ASIC3 subunit. FIG. 9Ashows the effects of the C-terminal fragments of ASIC3 (upper traces)and ASIC2A (lower traces). FIG. 9B shows the effects of the N-terminalfragments of ASIC3 (upper traces) and ASIC2A (lower traces).

FIG. 10 reveals the selective modulatory effect of 10 μM NPFF on theproton-gated cationic currents evoked by human heteromeric ASICreceptors expressed in Xenopus oocytes . A. Human homomeric ASIC2A,ASIC3 and heteromeric ASIC2S.2 (ASIC2A+3) response to pH 4 application(bar). B. Same as in A but in the presence of NPFF. Note the change indesensitization kinetics in presence of the peptide and the potentiationof the current mediated by heteromeric ASIC2S.2. C. Quantitative effectsof NPFF on peak currents evoked by acidic pH for the three subtypes ofhuman ASICs. Values expressed as % of control are mean±SEM

FIG. 11 shows the modulatory effect of 10 μM NPFF on the proton-gatedcationic currents evoked by rat homomeric ASIC3 and heteromeric ASIC2S.2(ASIC2A+3) receptors expressed in Xenopus oocytes . A. rat homomericASIC2A, ASIC3 and heteromeric ASIC2S.2 response to pH 4 application(bar). B. Same as in A but in the presence of NPFF. Note the morepronounced potentiation of the current mediated by heteromeric ASIC2S.2than by homomeric ASIC3. C. Histograms illustrating the effects of NPFFon peak currents evoked by acidic pH for the three subtypes of ratASICs. Values expressed as % of control are mean±SEM.

FIG. 12 Dose-response curves of NPFF (A) and FMRFamide (B) on thepotentiation of currents induced by pH 4 on human ASIC2S.2 (ASIC2A+3)receptors. NPFF was found more potent (EC50=2 μM, n=6) than FMRFamide(EC50=13 μM, n=12) in modulating ASIC2S.2-mediated cation currents.Values are expressed as mean±SEM and represent % of response to pH 4measured in presence of 10-8 M peptide.

FIG. 13A. NPFF potentiated the response of human ASIC2S.2 (ASIC2A+3)receptors to pH 6 stimulation. B. pH dose-response curves of humanheteromeric ASIC2S.2 (ASIC2A+3) receptors in the presence or absence ofneuropeptides. Sensitivity and maximal response to acidificationincreased in presence of 100 μM FMRFamide and the effects were evengreater in the presence of 10 μM NPFF. Reference value for currentnormalization corresponded to maximal control currents induced at pH 3.Values are expressed as mean±SEM.

DETAILED DESCRIPTION OF THE INVENTION

Preambule

It is understood that the present invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahost cell” includes a plurality of such host cells; reference to “theantibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

Definitions

For the sake of clarity, FIG. 8 summarises the nomenclature of the ASICsubunits encompassed by the present invention and will be applied toidentify without ambiguity the ASIC subunits herein referred to. Unlessspecified otherwise, reference to an ASIC subunit is not intended to belimited to a particular species but includes the orthologues orhomologues of any species.

“Polynucleotide” and “nucleic acids” as used herein refers to single- ordouble-stranded molecules which may be “deoxyribonucleic acid” (DNA),comprised of the nucleotide bases A, T, C and G, or “ribonucleic acid”(RNA), comprised of bases A, U (substitutes for T), C and G.Polynucleotides may represent a coding strand or its complement, thesense or anti-sense strands. Polynucleotides may be identical insequence to the sequence which is naturally occurring or may includealternative codons which encode the same amino acid as that which isfound in the naturally occurring sequence (Lewin: “Genes V”, Chapter 7;Oxford University Press, 1994). Furthermore, polynucleotides may includecodons which represent conservative substitutions of amino acids. Theterm “polynucleotide” will also include all possible alternate forms ofDNA or RNA, such as genomic DNA (both introns and exons), complementaryDNA (cDNA), cRNA, messenger RNA (mRNA), and DNA or RNA prepared bypartial or total chemical synthesis from nucleotide bases, includingmodified bases, such as tritylated bases and unusual bases such asinosine. Polynucleotides will also embrace all chemically, enzymaticallyor metabolically modified forms of DNA or RNA, as well as the chemicalforms of DNA and RNA characteristic of viruses.

The term “oligonucleotide” or “oligo” will refer short polynucleotides,typically between 10 to 40 bases in length.

“Polypeptide” as used herein refers to a molecule comprised of two ormore amino acids joined to each other by peptide bonds or modifiedpeptide bonds (i.e. isosteres). Amino acids include all 20 naturallygene-encoded amino acids as well as naturally or chemically modifiedamino acids. Polypeptides refer to both short chains of amino acids,commonly referred to as peptides, oligopeptides, or oligomers, and tolonger chains, commonly referred to as proteins. Thus, “amino acidsequence” as used herein refers to an oligopeptide, peptide,polypeptide, or protein molecule and fragments or portions thereof,corresponding to a naturally occurring or synthetic molecule. Where“amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule. Furthermore,polypeptides will also include amino acid sequences modified either bynatural processes, such as posttranslational processing, or by chemicalmodification techniques, which are well known in the art. A givenpolypeptide may contain many types of modifications or a givenmodification may be present in the same or varying degrees at severalsites in a given polypeptide. Modifications can occur anywhere in thepolypeptide, including but not limited to, the peptide backbone, theamino acid side-chains and the amino or carboxyl termini. All the abovereferred to modifications as well as their practice are well describedin the research literature, both in basic texts and detailed monographs(“Proteins: Structure and Molecular Properties”; Creighton T E, FreemanW H, 2^(nd) Ed., New-York, 1993; “Posttranslational CovalentModification of Proteins”, Johnson B C, ed., Academic Press, New-York,1983; Also: Seiter et al., Meth Enzymol 1990; 182: 626, and Rattan etal., Ann NY Acad Sci 1992; 663: 48).

“Peptide nucleic acid”, as used herein, refers to a molecule whichcomprises an oligonucleotide to which an amino acid residue, such aslysine, and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen et al. AnticancerDrug Des 1993; 8: 53).

ASIC2A or ASIC3, as used herein, refers to the amino acid sequences ofsubstantially purified ASIC2A and ASIC3 obtained preferably but notexclusively from human or rat, from any source whether natural,synthetic, semi-synthetic, or recombinant.

The term “variant” as used herein is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptide,respectively. A typical variant of a polynucleotide differs innucleotide sequence from another reference polynucleotide. Differencesin the nucleotide sequence of the variant may or may not alter the aminoacid sequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,insertions, deletions, fusions, and truncations in the polypeptideencoded by the reference sequence, as discussed below. A typical variantof a polypeptide differs in amino acid sequence from another referencepolypeptide. Generally, differences are such that the sequences of thereference polypeptide and the variant are closely similar overall and,in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,insertions or deletions in any combination. A substituted or insertedamino acid residue may or may not be one encoded by the genetic code. Avariant of a polynucleotide or polypeptide may be naturally occurringsuch as an allelic or a pseudoallelic variant, including polymorphismsor mutations at on or more bases, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniquesor by direct synthesis. The term “mutant” are encompassed by the abovedefinition of non-natural variants.

“splice variants” as referred to herein are variants, which result fromthe differential or alternative splicing and assembly of exons presentin a given gene. Typically, the encoded proteins will display totalidentity in most regions, but lower identity in the specific regionencoded by different exons.

A “deletion”, as used herein, refers to a change in either amino acid ornucleotide sequence in which one or more amino acids or nucleotideresidues, respectively, are absent, as compared to a referencepolypeptide or polynucleotide.

An “insertion” or “addition”, as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to areference polypeptide or polynucleotide.

A “substitution”, as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively, as compared to a reference polypeptide or polynucleotide.

The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding ASIC2A or ASIC3 or the encodedASIC2A or ASIC3. Illustrative of such modifications would be replacementof hydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativewould encode a polypeptide which may or may not retain some or all ofthe essential biological characteristics of the natural molecule.

The term “identity” as used herein refers to a measure of the extent ofidentical nucleotides or amino acids that two or more polynucleotide oramino acid sequences have in common. In general, the sequences arealigned so that the highest order match is obtained, referred to as the“alignment”. Such optimal alignments make use of gaps, which areinserted to maximize the number of matches using local homologyalgorithms, such as the Smith-Waterman alignment. The terms “identity”,or “similarity”, or “homology”, or “alignment” are well known to skilledartisans and methods to perform alignments and measure identity arewidely described and taught in the literature: Dayhoff et al., MethEnzymol 1983; 91: 524—Lipman D J and Pearson W R, Science 1985; 227:1435—Altschul et al., J Mol Biol 1990; 215: 403.—Pearson W R, Genomics1991; 11: 635.—Gribskov M and Devreux J, eds. (1992) Sequence AnalysisPrimer, W H Freeman & Cie, New-York.—Altschul et al., Nature Gen 1994;6: 119. Furthermore, methods to perform alignments and to determineidentity and similarity are codified in computer programs and softwarepackages, some of which may also be web-based and accessible on theinternet. Preferred software include but are not limited to BLAST (BasicLocal Alignment Search Tools), including Blastn, Blastp, Blastx, tBlastn(Altschul et al., J Mol Biol 1990; 215: 403), FastA and TfastA (Pearsonand Lipman, PNAS 1988; 85: 2444), Lasergene99 (DNASTAR, Madison Wis.),Omiga 2.0 or MacVector (Oxford Molecular Group, Cambridge, UK),Wisconsin Package (Genetic Computer Group (GCG), Madison, Wis.), VectorNTI Suite (InforMax Inc., N. Bethesta, Md.), GeneJockey II (Biosoft,Cambridge, UK).

As an illustration, by a polynucleotide having a nucleotide sequencewith at least, for example, 95% “identity” to a reference nucleotidesequence of SEQ ID NO: 1, is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations, ordivergent nucleotides, per 100 nucleotides of the reference nucleotidesequence of SEQ ID NO: 1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5′ or 3′ terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more continuous groups within thereference sequence.

Similarly, by a polypeptide having an amino acid sequence having atleast, for example, 95% “identity” to a reference amino acid sequence ofSEQ ID NO: 2, is intended that the amino acid sequece of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid sequence of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence, or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more continuous groups within thereference sequence.

The term “biologically active” or “biological activity”, as used herein,refer to a protein having structural, regulatory, biochemical,electrophysiological or cellular functions of a naturally occurringmolecule. Likewise, “immunologically active” refers to the capability ofthe natural, recombinant, or synthetic ASIC2A, or ASIC3, or ASIC-2S.2,or any oligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

As used herein, “specific antibodies” “antibodies to ASIC-2S.2” and“ASIC-2S.2-specific antibodies” refer to antibodies which specificallybind the ASIC-2S.2 protein complex, or specifically bind ASIC2A and/orASIC3 in their heteromultimeric conformation, or antibodies specificallybinding ASIC2A or ASIC3 and capable of inhibiting, preventing ordisrupting the interaction, association or assembly between ASIC2A andASIC3, as described herein.

As used herein, “proton-gated” and “acid-sensing” refer to an increasein cation permeability of a channel molecule induced by an increase inproton ion concentration, also described as increased acidity orlowering of pH.

As used herein, “gain of function” refers to a potentiation of anexisting biological activity and/or an acquisition of a novel biologicalactivity. Similarly, “Loss of function” refers to a partial or completeloss of one or more existing biological activities. The expression“Dominant-negative” refers to a loss-of-function derivative of ASIC2A orASIC3, which when coexpressed with a fully functional ASIC2A or ASIC3,in vivo, for example as a transgene, or in vitro, for example in anassay used to test the specific biological activity (for example“acid-sensing”), will dominate the response and impose the loss ofbiological activity on all other ASIC subunits associated with it.

The term “agonist”, as used herein, refers to a molecule, which causes achange in ASIC-2S.2 and modulates or induces directly or indirectly abiological activity of the ASIC-2S.2 heteromultimeric channels. Agonistsmay include proteins, nucleic acids, aptamers, carbohydrates, or anyother molecules, which display the properties described herein above.

The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which modulates or blocks directly or indirectly a biologicalactivity of the ASIC-2S.2 heteromultimeric channels. Antagonists andinhibitors may include proteins, nucleic acids, aptamers, carbohydrates,or any other molecules which display the properties described hereinabove.

The term “modulate”, as used herein, refers to a change or an alterationin the biological activity of ASIC-2S.2 heteromultimeric channels.Modulation may be an increase or a decrease in protein activity, achange in binding characteristics, or any other change in thebiological, functional or immunological properties of the ASIC-2S.2.

The term “mimetic”, as used herein, refers to a molecule, the structureof which is developed from knowledge of the structure of ASIC2A or ASIC3or portions thereof and, as such, is able to effect some or all of theactions of ASIC-like molecules.

The term “substantially purified”, as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,more preferably 90%, even more preferable 95%, and most preferably 99%free from other components with which they are naturally associated.

“Amplification” as used herein refers to the production of additionalcopies of a nucleic acid and is generally carried out using polymerasechain reaction (PCR) technologies well known in the art (“PCR Primer: alaboratory manual” Dieffenbach C W and Dveksler G S, eds., 1995, CSHLPress, Plainview, N.Y.).

The term “hybridization”, as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term “hybridization complex”, as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., RNAse ProtectionAssay analysis) or between one nucleic acid sequence present in solutionand another nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

The terms “complementary” or “complementarity”, as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single-stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acid strands.

The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

As known in the art, numerous equivalent conditions may be employed tocomprise either low or high stringency conditions. Factors such as thelength and nature (DNA, RNA, base composition) of the sequence, natureof the target (DNA, RNA, base composition, presence in solution orimmobilization, etc.), and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfateand/or polyethylene glycol) are considered and the hybridizationsolution may be varied to generate conditions of either low or highstringency different from, but equivalent to, the above listedconditions.

The term “stringent conditions”, as used herein, is the “stringency”which occurs within a range from about Tm-5° C. (5° C. below the meltingtemperature (Tm) of the probe) to about 20° C. to 25° C. below Tm. Aswill be understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences.

The term “antisense”, as used herein, refers to nucleotide sequenceswhich are complementary to a specific DNA or RNA sequence. The term“antisense strand” is used in reference to a nucleic acid strand that iscomplementary to the “sense” strand. Antisense molecules may be producedby any method, including synthesis by ligating the gene(s) of interestin a reverse orientation to a viral promoter, which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designation “negative” is sometimes used in reference tothe antisense strand, and “positive” is sometimes used in reference tothe sense strand.

The term “portion”, as used herein, with regard to a protein (as in “aportion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein “comprising atleast a portion of the amino acid sequence of SEQ ID NO:2” encompassesthe full-length human ASIC2A and fragments thereof.

“Transformation” or “transfection”, as defined herein, describes aprocess by which exogenous DNA enters and changes a recipient cell. Itmay occur under natural or artificial conditions using various methodswell known in the art. Transformation or transfection may rely on anyknown method for the insertion of foreign nucleic acid sequences into aprokaryotic or eukaryotic host cell. The method is selected based on thehost cell being transformed and may include, but is not limited to,viral infection, electroporation, lipofection, and particle bombardment.Such “transformed” or “transfected” cells include stably transformedcells in which the inserted DNA is capable of replication either as anautonomously replicating plasmid or as part of the host chromosome. Theyalso include cells that transiently express the inserted DNA or RNA forlimited periods of time.

The term “antigenic determinant”, as used herein, refers to that portionof a molecule that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody and a protein or peptide,mean that the interaction is dependent upon the presence of a particularstructure (i.e., the antigenic determinant or epitope) on the protein;in other words, the antibody is recognizing and binding to a specificprotein structure rather than to proteins in general. For example, if anantibody is specific for epitope “A”, the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled “A” and the antibody will reduce the amount of labeled A boundto the antibody.

The term “sample”, as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acids encoding ASIC2Aand ASIC3 or fragments thereof may comprise a cell, chromosomes isolatedfrom a cell (e.g., a spread of metaphase chromosomes), genomic DNA (insolution or bound to a solid support such as for Southern analysis), RNA(in solution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

The term “correlates with expression of a polynucleotide”, as usedherein, indicates that the detection by northern analysis and/or RT-PCRof the presence of ribonucleic acid that is related to SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5 or SEQ ID NO:7 is indicative of the presence ofmRNA encoding, respectively, hASIC2A, hASIC3, rASIC2A or rASIC3 in asample and thereby correlates with expression of the transcript encodingthe protein.

“Alterations” in the polynucleotides of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5 or SEQ ID NO:7, as used herein, comprise any alteration in thesequence of polynucleotides encoding, respectively, hASIC2A, hASIC3,rASIC2A or rASIC3, including deletions, insertions, and point mutationsthat may be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genomic DNA sequencewhich encodes hASIC2A, hASIC3, rASIC2A or rASIC3 (e.g., by alterationsin the pattern of restriction fragment length polymorphisms capable ofhybridizing to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7),the inability of a selected fragment of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5 or SEQ ID NO:7 to hybridize to a sample of genomic DNA (e.g., usingallele-specific oligonucleotide probes), and improper or unexpectedhybridization, such as hybridization to a locus other than the normalchromosomal locus for the polynucleotide sequence encoding hASIC2A,hASIC3, rASIC2A or rASIC3 (e.g., using fluorescent in situ hybridization(FISH) to metaphase chromosome spreads).

As used herein, the term “antibody” refers to intact molecules as wellas fragments thereof, such as Fa, F(ab′)₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind hASIC2A, hASIC3,rASIC2A or rASIC3 polypeptides can be prepared using intact polypeptidesor fragments containing small peptides of interest as the immunizingantigen. The polypeptide or peptide used to immunize an animal can bederived from translated RNA or synthesized chemically, and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat or a rabbit). These methods are well described inthe literature: e.g. “Antobodies: A Laboratory Manual”, Harlow E andLane D, eds., 1998, CSHL Press, Plainview, N.Y.).

The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

DISCLOSURE OF THE INVENTION

The present invention is based on the discovery of a novel Acid SensingIon Channel (ASIC) with distinctive channel properties and biologicalactivity. This novel channel, ASIC-2S.2 is a heteromultimeric complexcomprised of two different types of ASIC subunits, namely ASIC2A andASIC3 (see FIG. 8). The present invention is also based on the discoveryof a new use of polynucleotides encoding ASIC2A and ASIC3, said usebeing the inclusion of ASIC2A and ASIC3 in the assembly of aheteromultimeric ion channel. This invention further includes the use ofthe above compositions for diagnosis, prevention, or treatment ofdiseases related to the expression of the heteromultimeric ASIC channeldisclosed herein. The preferred polynucleotides are those of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7, encoding, respectively,the hASIC2A, hASIC3, rASIC2A and rASIC3 of SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6 and SEQ ID NO:8. The above enumerated polynucleotides havealready been embodied in the following patents or patent applications:U.S. Pat. No. 5,892,018; WO9835034; WO9921981; WO9854316. The generalnotion of heteromultimeric associations between subunits of the samefamily is widely recognized by those skilled in the art. The inventivestep resides in finding the functional combinations of known subunits aswell as the changes in channel properties and/or biological activity.Patent application WO9835034 does indeed claim hybrid ASIC channels butsupports this claim with indirect evidence suggesting interactionsbetween ASIC and MDEG1, MDEG1 and MDEG2, and between MDEG2 and DRASIC(see above). The present invention demonstrates for the first time thedirect biochemical interaction between two distinct ASIC subunits,namely ASIC2A and ASIC3, which produces a novel proton-gated ion channelwith distinctive properties.

The invention also encompasses heteromultimeric ASIC channels comprisedof ASIC2A and/or ASIC3 variants, in any possible combination ofwild-type and variant forms. A preferred ASIC2A variant is one having atleast 80%, and more preferably 90%, amino acid sequence identity to theASIC2A amino acid sequence of SEQ ID NO: 2 or SEQ ID NO.6. A mostpreferred ASIC2A variant is one having at least 95% amino acid sequenceidentity to SEQ ID NO: 2 or SEQ ID NO:6, while those with 97-99% aminoacid sequence identity are most highly preferred. A preferred ASIC3variant is one having at least 80%, and more preferably 90%, amino acidsequence identity to the ASIC3 amino acid sequence of SEQ ID NO:4 or SEQID NO.8. A most preferred ASIC3 variant is one having at least 95% aminoacid sequence identity to SEQ ID NO:4 or SEQ ID NO:8, while those with97-99% amino acid sequence identity are most highly preferred.

The invention also encompasses polynucleotides which encode ASIC2A orASIC3 polypeptides. Accordingly, any nucleic acid sequence, whichencodes the amino acid sequence of ASIC2A or ASIC3 can be used togenerate recombinant molecules which express ASIC2A or ASIC3. In aparticular embodiment, the invention encompasses the polynucleotidecomprising the nucleic acid of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5,and SEQ ID NO:7.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding ASIC2A or ASIC3, some bearing minimal homology to thenucleotide sequences of any known and naturally occurring gene, may beproduced. Thus, the invention contemplates each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequence of naturally occurring ASIC2A or ASIC3, and allsuch variations are to be considered as being specifically encompassed.

Although nucleotide sequences which encode ASIC2A or ASIC3 and theirvariants are preferably capable of hybridizing to the nucleotidesequence of the naturally occurring ASIC2A or ASIC3 under appropriatelyselected conditions of stringency, it may be advantageous to producenucleotide sequences encoding ASIC2A or ASIC3 or their derivativespossessing a substantially different or non-naturally occurring codonusage. Codons may be selected to increase the rate at which expressionof the peptide occurs in a particular prokaryotic or eukaryoticexpression host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding ASIC2A or ASIC3 and theirderivatives without altering the encoded amino acid sequences includethe production of RNA transcripts having more desirable properties, suchas a greater half-life, than transcripts produced from the naturallyoccurring sequence.

The invention also encompasses production of a DNA sequence, or portionsthereof, which encode ASIC2A or ASIC3 and their derivatives, entirely bysynthetic chemistry. After production, the synthetic gene may beinserted into any of the many available DNA vectors and cell systemsusing reagents that are well known in the art at the time of the filingof this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding ASIC2A or ASIC3 or anyportion thereof.

Further encompassed by the invention are polynucleotide sequences thatare capable of hybridizing to the claimed nucleotide sequences, and inparticular, to those shown in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5,and SEQ ID NO:7, under various conditions of stringency. Hybridizationconditions are based on the melting temperature (Tm) of the nucleic acidbinding complex or probe, as taught in Berger and Kimmel (Meth Enzymol1987: 152), and may be used at a defined stringency. Excluded from theabove defined polynucleotides are polynucleotides disclosed in PatentApplication WO9835034 under SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:6.

Altered nucleic acid sequences encoding ASIC2A or ASIC3 which areencompassed by the invention include deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent ASIC2A or ASIC3. Theencoded protein may also contain deletions, insertions, or substitutionsof amino acid residues, which result in a functionally equivalent ASIC2Aor ASIC3. Also encompassed by the invention are altered nucleic acidsequences, including deletions, insertions or substitutions, whichresult in a polynucleotide that encodes an ASIC2A or ASIC3 polypeptidewith increased or novel biological activity (“gain of function”), or anASIC2A or ASIC3 polypeptide with decreased or suppressed biologicalactivity (“Loss of function” or “Dominant-negative”). The encodedprotein may also contain deletions, insertions, or substitutions ofamino acid residues, which result in a functionally divergent ASIC2A orASIC3, as described herein above. Deliberate amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the biological activity of ASIC2A or ASIC3 isretained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid; positively charged amino acids mayinclude lysine and arginine; and amino acids with uncharged polar headgroups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; phenylalanine and tyrosine.

Also included within the scope of the present invention are alleles ofthe genes encoding ASIC2A or ASIC3. As used herein, an “allele” or“allelic sequence” is an alternative form of the gene, which may resultfrom at least one mutation in the nucleic acid sequence. Alleles mayresult in altered mRNAs or polypeptides whose structure or function mayor may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes, which give rise to alleles,are generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

Methods for DNA sequencing, which are well known and generally availablein the art, may be used to practice any embodiments of the invention.The methods may employ such enzymes as the Klenow fragment of DNApolymerase I, Sequenase II (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproofreading exonucleases such as the ELONGASE Amplification Systemmarketed by Gibco BRL (Gaithersburg, Md.) or the EXPAND High fidelity orLong-Template systems (Roche). Preferably, the process is automated withmachines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.),Peltier Thermal Cycler (PTC200; M.J. Research, Watertown, Mass.) and theABI 377 DNA sequencers (Perkin Elmer), to name a few.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devisecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. Genotyper™ and Sequence Navigator™,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter the ASIC2A orASIC3 coding sequence for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequence. For example, site-directedmutagenesis may be used to insert new restriction sites, to alterglycosylation patterns, to change codon preference, to produce splicevariants, or other mutations, and so forth. Alternatively, thenucleotide sequences can be engineered to generate chimeric ASIC2A orASIC3 channels, where portions of the ASIC2A channel are substitutedwith equivalent portions of the ASIC3 subunit and/or vice versa.Chimeric ASIC2A or ASIC3 can also be constructed by substitutingportions ASIC2A or ASIC3 with equivalent portions from other ASICsubunits, for example the ASIC1A or ASIC4. Nucleic acids can also beengineered to encode as a single polypeptide two or more ASIC2A and/orASIC3 subunits in tandem.

In another embodiment of the invention, a natural, modified, orrecombinant polynucleotide encoding ASIC2A or ASIC3 may be ligated to aheterologous sequence to encode a fusion protein. For example, toprovide biochemical evidence of direct protein-protein interactionsbetween ASIC2A and ASIC3, polynucleotides encoding ASIC2A and ASIC3 aremodified to encode chimeric ASIC2A or ASIC3 proteins with N- orC-terminal extensions adding a foreign epitope recognised bycommercially available antibodies or affinity resins. A fusion proteinmay also be engineered to contain a cleavage site located between theASIC2A or ASIC3 encoding sequence and the heterologous protein sequence,so that ASIC2A or ASIC3 may be cleaved and purified away from theheterologous moiety.

In another embodiment, the coding sequence of ASIC2A and/or ASIC3 may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers et al., Nuc. Acids Res. Symp. Ser. 1980; 215-23;Horn et al., Nuc. Acids Res. Symp. Ser. 1980; 225-232). Alternatively,the protein itself may be produced using chemical methods to synthesizethe ASIC2A and/or ASIC3 amino acid sequence, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge et al., Science 1995; 269: 202) and automatedsynthesis may be achieved, for example, using the ABI 431A PeptideSynthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton T.(1983) “Proteins, Structures and Molecular Principles”, W. H. Freeman &Co., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton T (1983), supra). Additionally, theamino acid sequence of ASIC2A and/or ASIC3, or any part thereof, may bealtered during direct synthesis and/or combined using chemical methodswith sequences from other proteins, or any part thereof, to produce avariant polypeptide.

In order to express a biologically active ASIC2A and/or ASIC3, thenucleotide sequence encoding ASIC2A and/or ASIC3 or functionalequivalents thereof, may be inserted into an appropriate expressionvector, i.e., a vector, which contains the necessary elements for thetranscription and translation of the inserted coding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing a ASIC2A and/or ASIC3 codingsequences and appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described in “Molecular Cloning: A Laboratory Manual”, Sambrook J,Ed., CSHL Press, 1989, Cold Spring Harbor, N.Y., and “Current Protocolsin Molecular Biology”, Ausubel et al., John Wiley & Sons, 1989, NewYork, N.Y.

A variety of expression vector/host systems may be utilized to containand express ASIC2A and/or ASIC3 coding sequences. These include, but arenot limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems.

The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla, Calif.) or pSport1™ plasmid (Gibco BRL) and ptrp-lac hybrids, andthe like may be used. Other preferred prokaryotic vectors include butare not limited to pQE-9, pQE60, pQE70 (Quiagen), pNH8A, pNH16a, pNH18a,pNH46A (Stratagene) ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). The baculovirus polyhedrin promoter may be used in insectcells. Promoters or enhancers derived from the genomes of plant cells(e.g., heat shock, RUBISCO; and storage protein genes) or from plantviruses (e.g., viral promoters or leader sequences) may be cloned intothe vector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding ASIC2Aand/or ASIC3, vectors based on SV40 or EBV may be used with anappropriate selectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for ASIC2A and/or ASIC3. For example,when large quantities of ASIC2A and/or ASIC3 are needed for theinduction of antibodies, vectors, which direct high level expression offusion proteins that are readily purified, may be used. Such vectorsinclude, but are not limited to, the multifunctional E. coli cloning andexpression vectors such as Bluescript® (Stratagene, La Jolla, Calif.),into which the sequence encoding ASIC2A or ASIC3 may be ligated in framewith sequences for the amino-terminal Methionine and the subsequent 7residues of β-galactosidase so that a hybrid protein is produced; pINvectors (Van Heeke and Schuster, J. Biol. Chem. 1989; 264: 5503); andthe like; pGEX vectors (Promega, Madison, Wis.) may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems may be designed to includeheparin, thrombin, or factor XA protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

In addition to bacteria, eucaryotic microbes, such as yeast, may also beused as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker'syeast, are most used although a number of other strains or species arecommonly available. Vectors employing, for example, the 2μ origin ofreplication of Broach et al. (Meth Enzymol 1983; 101: 307), or otheryeast compatible origins of replication (see, for example, Stinchcomb etal. Nature 1979: 282; 39, Tschumper et al., Gene 1980: 10; 157, Clarkeet al., Meth Enzymol 1983; 101: 300) may be used. Control sequences foryeast vectors include promoters for the synthesis of glycolytic enzymes(Hess et al. J Adv Enzyme Reg 1968; 7: 149; Holland et al., Biochemistry1978; 17: 4900). Additional promoters known in the art include thepromoter for 3-phosphoglycerate kinase (Hitzeman et al., J Biol Chem1980; 255: 2073), alcohol oxidase, and PGH. Other promoters, which havethe additional advantage of transcription controlled by growthconditions and/or genetic background are the promoter regions foralcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradativeenzymes associated with nitrogen metabolism, the alpha-factor system andenzymes responsible for maltose and galactose utilization. It is alsobelieved terminator sequences are desirable at the 3′ end of the codingsequences. Such terminators are found in the 3′ untranslated regionfollowing the coding sequences in yeast-derived genes. For reviews, see“Current Protocols in Molecular Biology”, Ausubel et al., John Wiley &Sons, 1989, New York, N.Y. and Grant et al., Meth Enzymol. 1987; 153:516.

In cases where plant expression vectors are used, the expression of asequence encoding ASIC2A or ASIC3 may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu et al., EMBO J. 1987; 6: 307;Brisson et al., Nature 1984; 310: 511). Alternatively, plant promoterssuch as the small subunit of RUBISCO or heat shock promoters may be used(Coruzzi et al., EMBO J. 1984; 3: 1671; Broglie et al., Science 1984;224: 838; Winter et al., Results Probl. Cell Differ 1991; 17: 85). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs S or Murry L E in “McGraw Hill Yearbook of Science and Technology”McGraw Hill, 1992, New York, N.Y.; pp. 191-196 or Weissbach andWeissbach in “Methods for Plant Molecular Biology”, Academic Press,1988, New York, N.Y.; pp. 421-463).

An insect system may also be used to express ASIC2A and/or ASIC3. Forexample, in one such system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequence encoding ASIC2Aand/or ASIC3 may be cloned into a non-essential region of the virus,such as the polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of ASIC2A or ASIC3 will render thepolyhedrin gene inactive and produce recombinant virus lacking coatprotein. The recombinant viruses may then be used to infect, forexample, S. frugiperda cells or Trichoplusia larvae in which ASIC2Aand/or ASIC3 may be expressed (Smith et al., J Virol 1983; 46: 584;Engelhard et al., Proc Natl Acad Sci 1994; 91: 3224).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a sequence encoding ASIC2A and/or ASIC3 may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable virus,which is capable of expressing ASIC2A and/or ASIC3 in infected hostcells (Logan and Shenk, Proc Natl Acad Sci 1984; 81: 3655). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of a sequence encoding ASIC2A and/or ASIC3. Such signalsinclude the ATG initiation codon and adjacent sequences. In cases wheresequences encoding ASIC2A or ASIC3, together with their initiationcodon, and upstream sequences are inserted into the appropriateexpression vector, no additional transcriptional or translationalcontrol signals may be needed. However, in cases where only codingsequence, or a portion thereof, is inserted, exogenous translationalcontrol signals including the ATG initiation codon should be provided.Furthermore, the initiation codon should be in the correct reading frameto ensure the correct translation of the entire insert. Exogenoustranslational elements and initiation codons may be of various origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used, such as those described in the literature(Scharf et al., Results Probl Cell Differ 1994; 20: 125; Bittner et al.,Meth Enzymol 1987; 153: 516).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, WI38,and COS, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

In a preferred expression system, cDNAs or cRNAs encoding ASIC2A and/orASIC3 are coinjected directly into Xenopus oocytes , cDNAs into nucleiand cRNA into the cytoplasm, thereby allowing for in vitro translationand assembly into a functional heteromultimeric proton-gated channelcapable of demonstrating functional characteristics of nativeproton-gated channels including ion selectivity, gating-kinetics, ligandpreferences, and sensitivity to pharmacological agents such as amiloridefor a model assay which mimics in vivo characteristics.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines, which stably expressASIC2A or ASIC3, or both ASIC2A and ASIC3, may be transformed usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor separate vector. Following the introduction of the vector, cells maybe allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells, which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., Cell 1977; 11: 223) and adeninephospho-ribosyltransferase (Lowy et al., Cell 1980; 22: 817) genes whichcan be employed in tk+- or aprt+-cells, respectively. Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection; for example, dhfr, which confers resistance tomethotrexate (Wigler et al., Proc Natl Acad Sci 1980; 77: 3567); npt,which confers resistance to the aminoglycosides neomycin and G-418(Colbere-Garapin et al., J Mol Biol 1981; 150: 1) and als or pat, whichconfer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry L E, supra). Additionalselectable genes have been described, for example, trpB, which allowscells to utilize indole in place of tryptophan, or hisD, which allowscells to utilize histinol in place of histidine (Hartman and Mulligan,Proc Natl Acad Sci 1988; 85: 8047). Recently, the use of visible markershas gained popularity with such markers as anthocyanins, β-glucuronidaseand its substrate GUS, and luciferase and its substrate luciferin, beingwidely used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system (Rhodes et al., Methods Mol Biol 1995; 55: 121).

Although the presence/absence of marker for gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding ASIC2Aand/or ASIC3 is inserted within a marker gene sequence, recombinantcells containing sequences encoding ASIC2A and/or ASIC3 can beidentified by the absence of marker gene function. Alternatively, amarker gene can be placed in tandem with a sequence encoding ASIC2A orASIC3 under the control of a single promoter. Expression of the markergene in response to induction or selection usually indicates expressionof the tandem gene as well.

Alternatively, host cells, which contain the coding sequences for ASIC2Aand/or ASIC3 and express both ASIC2A and ASIC3 may be identified by avariety of procedures known to those of skill in the art. Theseprocedures include, but are not limited to, DNA-DNA or DNA-RNAhybridizations and protein bioassay or immunoassay techniques, whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of the nucleic acid or protein.

The presence of the polynucleotide sequences encoding ASIC2A and/orASIC3 can be detected by DNA-DNA or DNA-RNA hybridization oramplification using probes or portions or fragments of polynucleotidesencoding ASIC2A or ASIC3. Nucleic acid amplification based assaysinvolve the use of oligonucleotides or oligomers based on the ASIC2A- orASIC3-encoding sequences to detect transformants containing DNA or RNAencoding ASIC2A and/or ASIC3. As used herein “oligonucleotides” or“oligomers” refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

A variety of protocols for detecting and measuring the co-expression ofASIC2A and ASIC3, using either polyclonal or monoclonal antibodiesspecific for each protein are known in the art. Examples includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), andfluorescent activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on ASIC2A or ASIC3 is preferred, but acompetitive binding assay may be employed. These and other assays aredescribed, among other places, in “Serological Methods: A LaboratoryManual”, Hampton et al., APS Press, 1990, St-Paul, Mich. and Maddox etal., J Exp Med 1983; 158: 1211).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding ASIC2A or ASIC3include oligo-labeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequenceencoding ASIC2A or ASIC3, or any portion thereof, may be cloned into avector for the production of an mRNA probe. Such vectors are known inthe art, are commercially available, and may be used to synthesize RNAprobes in vitro by addition of an appropriate RNA polymerase such as T7,T3 or SP6 and labeled nucleotides. These procedures may be conductedusing a variety of commercially available kits: from e.g. Pharmacia &Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. BiochemicalCorp. (Cleveland, Ohio), or Ambion (Austin, Tex.). Suitable reportermolecules or labels, which may be used, include radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells co-transformed with nucleotide sequences encoding both ASIC2Aand/or ASIC3 may be cultured under conditions suitable for theexpression and recovery of the proteins from cell culture. The proteinsproduced by a recombinant cell may be secreted or containedintracellularly depending on the sequence and/or the vector used. Aswill be understood by those of skill in the art, expression vectorscontaining polynucleotides, which encode ASIC2A or ASIC3 may be designedto contain signal sequences which direct secretion of ASIC2A and/orASIC3 through a prokaryotic or eukaryotic cell membrane.

Other recombinant constructions may be used to join sequences encodingASIC2A or ASIC3 to nucleotide sequence encoding a polypeptide domain,which will facilitate purification of the expressed proteins. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp.,Seattle, Wash.). The inclusion of cleavable linker sequences such asthose specific for Factor XA or enterokinase (Invitrogen, San Diego,Calif.) between the purification domain and ASIC2A or ASIC3 may be usedto facilitate purification. One such expression vector provides forexpression of a fusion protein containing ASIC2A and/or ASIC3, athioredoxin or an enterokinase cleavage site, and followed by sixhistidine residues. The histidine residues facilitate purification onIMIAC (immobilized metal ion affinity chromatography as described inPorath et al., Prot Exp Purif 1992; 3: 263) while the enterokinasecleavage site provides a means for purifying ASIC2A or ASIC3 from thefusion protein. A discussion of vectors which contain fusion proteins isprovided in Kroll et al. (DNA Cell Biol 1993; 12: 441).

In addition to recombinant production, fragments of ASIC2A and/or ASIC3may be produced by direct peptide synthesis using solid-phase techniques(see Stewart et al., “Solid-Phase Peptide Synthesis”, W H Freeman & Co.,1969, San Francisco, Calif.; Merrifield et al., J Am Chem Soc 1963; 85:2149). Chemical synthesis may be performed using manual techniques or byautomation. Automated synthesis may be achieved, for example, usingApplied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Variousfragments of ASIC2A and/or ASIC3 may be chemically synthesizedseparately and combined using chemical methods to produce thefull-length molecule.

Thus as set forth herein the invention includes the provision of a novelsubfamily of heteromultimeric proton-gated ion channels as exemplifiedby the novel association of ASIC2A and ASIC3 polypeptides, encoded,respectively, by nucleic acids of SEQ ID NO: 1 or NO:5 and SEQ ID NO: 3or NO:7 as well as DNA sequences which hybridize thereto under stringenthybridization conditions, and DNA sequences encoding the same allelicvariant or analogue proton-gated channel protein through use of at leastin part degenerate codons. The ASIC-2S.2 channel complex can also beused to located and identify other closely related members of thissubfamily as described in Cannessa et al (Nature 1994; 367: 463).

Polypeptides of SEQ ID NO:2, NO:4, NO:6 and NO:8, as well as anyprotein, protein fragment, synthetic protein or peptide thereof areprojected to have uses earlier described including therapeutic,diagnostic, and prognostic assays and protocols and will provide thebasis for monoclonal and polyclonal antibodies specifically reactivewith the channel protein.

Therapeutics

In another embodiment of the invention, ASIC-2S.2 heteromultimericchannels may be used for therapeutic purposes. Based on the mRNAdistribution patterns of ASIC2A and ASIC3 showing that ASIC2A and ASIC3transcripts are primarily but not exclusively associated with cells ofthe peripheral and central nervous systems, ASIC-2S.2 is believed toplay a role in the regulation of neurotransmitter release, neuronalexcitability, or excitotoxicity. Indeed, secretory granules and synapticvesicules are known to contain high concentrations of protons (lowintravesicular pH), which are co-released with other neurotransmittersduring regulated and constitutive exocytosis. Released protons mightthus activate pre- and/or post-synaptic, or extrasynaptic ASIC-2S.2receptors. Indeed, under certain conditions, low pH or extracellularacidosis has been shown to influence synaptic transmission as well asthe induction of long-term potentiation (Igelmund et al., Brain Res1995; 689: 9; Velisek et al., Hippocampus 1998; 8: 24). Also, in certainanimal seizure models, neuroprotective effects of low pH have beenobserved (Velisek et al., Exp Brain Res 1994; 101: 44). Thus, animportant use of ASIC-2S.2 is screening for compounds that regulateneurotransmitter release, synaptic efficacy, neuroexcitability, orneurotoxicity. Such compounds may have utility in a number ofphysiological and pathological situations pertaining, for example, tocognition, perception, learning, memory, pain and many others. Morespecifically, in situ hybridization and duplex RT-PCR analysis (FIGS. 4and 5) indicate that coexpression of ASIC2A and ASIC3 is regionspecific. Interestingly, in contrast to what is reported for the rat, wereport herein for the first time that ASIC2A is highly expressed inhuman sensory ganglia, such as the trigeminal ganglia. Furthermore, thehighest probability of coexpression of ASIC2A and ASIC3 has also beenfound in these sensory ganglia. This strongly suggests an important rolefor the ASIC-2S.2 channels in pain and/or somato-sensory transmission.

In one embodiment, antagonists or inhibitors of the ASIC-2S.2 proteincomplex or vectors expressing antisense sequences may be used to treatdisorders and diseases of the nervous system resulting from altered iontransport, signal transmission, and apoptosis. Such diseases include,but are not limited to, chronic pain, neuropathic pain such asdiabetic-, cancer-, and AIDS-related, neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, Huntington's disease,Creutzfeld-Jacob disease, and amyotrophic lateral sclerosis, anddementias, including AIDS-related, as well as convulsions, epilepsy,stroke, and anxiety and depression.

In another embodiment, antagonists or inhibitors of the ASIC-2S.2protein complex or vectors expressing antisense sequences may be used totreat cardiovascular diseases such as angina, congestive heart failure,vasoconstriction, hypertension, atherosclerosis, restenosis, andbleeding.

Agonists, which enhance the activity and antagonists, which block ormodulate the effect of ASIC-2S.2 may be used in those situations wheresuch enhancement or inhibition is therapeutically desirable. Suchagonists, antagonists or inhibitors may be produced using methods, whichare generally known in the art, such as screening libraries ofpharmaceutical agents for compounds, which directly (or indirectly) andspecifically interact and/or bind ASIC-2S.2. Other methods involve theuse of purified ASIC2A and/or ASIC3 to produce antibodies. For example,in one aspect, antibodies which are specific for ASIC2A or ASIC3, orASIC-2S.2 may be used directly as an antagonist of ASIC-2S.2, orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue, which express ASIC-2S.2.

The antibodies may be generated using methods that are well known in theart. Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies, (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith ASIC2A, or ASIC3, or with ASIC2A and ASIC3, including covalentlylinked ASIC2A-ASIC3 tandems and ASIC2A-ASIC3 chimers, or any fragment oroligopeptide thereof which has immunogenic properties. Depending on thehost species, various adjuvants may be used to increase immunologicalresponse. Such adjuvants include, but are not limited to, Freund's,mineral gels such as aluminum hydroxide, and surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvantsused in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvumare especially preferable.

It is preferred that the peptides, fragments, or oligopeptides used toinduce antibodies to ASIC-2S.2 have an amino acid sequence consisting ofat least five amino acids, and more preferably at least 10 amino acids.It is also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of ASIC2A or ASIC3 amino acids may be fused with those ofanother protein such as keyhole limpet hemocyanin and antibody producedagainst the chimeric molecule.

Monoclonal antibodies to ASIC-2S.2 may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Koehler et al. Nature 1975; 256: 495; Kosbor etal., Immunol Today 1983; 4: 72; Cote et al., Proc Natl Acad Sci 1983;80: 2026; Cole et al., “Monoclonal Antibodies and Cancer Therapy”, AlanR. Liss Inc., 1985, New York, N.Y., pp. 77-96).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al. (1984) Proc. Natl.Acad. Sci. 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608;Takeda et al. (1985) Nature 314:452-454). Alternatively, techniquesdescribed for the production of single chain antibodies may be adapted,using methods known in the art, to produce ASIC-2S.2-specific singlechain antibodies. Antibodies with related specificity, but of distinctidiotypic composition, may be generated by chain shuffling from randomcombinatorial immunoglobin libraries (Burton, D. R. (1991) Proc. Natl.Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for ASIC-2S.2may also be generated. For example, such fragments include, but are notlimited to, the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse et al. (1989) Science 256:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between ASIC-2S.2 and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering ASIC-2S.2 epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingASIC2A and/or ASIC3, or any fragment thereof, or antisense sequences,may be used for therapeutic purposes. In one aspect, antisense to thepolynucleotide encoding ASIC2A and/or ASIC3 may be used in situations inwhich it would be desirable to block the synthesis of the ASIC-2S.2protein complex. In particular, cells may be transformed with sequencescomplementary to polynucleotides encoding ASIC2A and/or ASIC3. Thus,antisense sequences may be used to modulate ASIC-2S.2 activity, or toachieve regulation of gene function. Such technology is now well knownin the art, and sense or antisense oligomers or larger fragments, can bedesigned from various locations along the coding or control regions ofsequences encoding ASIC2A and/or ASIC3.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods, which are well known to those skilled in the art,can be used to construct recombinant vectors which will expressantisense polynucleotides of the genes encoding ASIC2A and/or ASIC3.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

Genes encoding ASIC2A and/or ASIC3 can be turned off by transforming acell or tissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes ASIC2A and/or ASIC3.Such constructs may be used to introduce untranslatable sense orantisense sequences into a cell. Even in the absence of integration intothe DNA, such vectors may continue to transcribe RNA molecules until allcopies are disabled by endogenous nucleases.

Transient expression may last for a month or more with a non-replicatingvector and even longer if appropriate replication elements are part ofthe vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA or PNA, to the control regionsof the gene encoding ASIC2A or ASIC3, i.e., the promoters, enhancers,and introns. Oligonucleotides derived from the transcription initiationsite, e.g., between positions −10 and +10 from the 5′ end of thetranscript, are preferred. Similarly, inhibition can be achieved using“triple helix” base-pairing methodology. Triple helix pairing is usefulbecause it causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and Carr, B. I. Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding ASIC2A and/or ASIC3.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of RNA molecules. Theseinclude techniques for chemically synthesizing oligonucleotides such assolid phase phosphoramidite chemical synthesis. Alternatively, RNAmolecules may be generated by in vitro and in vivo transcription of DNAsequences encoding ASIC2A and/or ASIC3. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the moleculeor the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysuitable subject including, for example, mammals such as dogs, cats,cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of ASIC-2S.2, or anycomponent thereof, antibodies to ASIC-2S.2, mimetics, agonists,antagonists, or inhibitors of ASIC-2S.2. The compositions may beadministered alone or in combination with at least one other agent, suchas stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances, which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of ASIC-2S.2, or any component thereof,such labeling would include amount, frequency, and method ofadministration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rats, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example ASIC-2S.2 or any component or fragment thereof,antibodies against ASIC-2S.2, agonists, antagonists or inhibitors ofASIC-2S.2, which ameliorates the symptoms or condition. Therapeuticefficacy and toxicity may be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED₅₀ (thedose therapeutically effective in 50% of the population) and LD₅₀ (thedose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can beexpressed as the ratio, LD₅₀/ED₅₀.

Pharmaceutical compositions, which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind ASIC-2S.2 maybe used for the diagnosis of conditions or diseases characterized byexpression of ASIC-2S.2, or in assays to monitor patients being treatedwith ASIC-2S.2 agonists, antagonists or inhibitors. The antibodiesuseful for diagnostic purposes may be prepared in the same manner asthose described above for therapeutics. Diagnostic assays for ASIC-2S.2include methods which utilize the antibody and a label to detectASIC-2S.2, or any component therof, in human body fluids or extracts ofcells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by joining them, either covalently ornon-covalently, with a reporter molecule. A wide variety of reportermolecules which are known in the art may be used, several of which aredescribed above.

A variety of protocols including ELISA, RIA, and FACS for measuringASIC-2S.2 are known in the art and provide a basis for diagnosingaltered or abnormal levels of ASIC-2S.2 expression. Normal or standardvalues for ASIC-2S.2 expression are established by combining body fluidsor cell extracts taken from normal mammalian subjects, preferably human,with antibody to ASIC-2S.2 under conditions suitable for complexformation. The amount of standard complex formation may be quantified byvarious methods, but preferably by photometric, means. Quantities ofASIC-2S.2 expressed in subject, control and disease, samples frombiopsied tissues are compared with the standard values. Deviationbetween standard and subject values establishes the parameters fordiagnosing disease.

In another embodiment of the invention, the polynucleotides encodingASIC-2S.2 may be used for diagnostic purposes. The polynucleotides whichmay be used include oligonucleotide sequences, antisense RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofASIC-2S.2 may be correlated with disease. The diagnostic assay may beused to distinguish between absence, presence, and excess expression ofASIC-2S.2, and to monitor regulation of ASIC-2S.2 levels duringtherapeutic intervention. The diagnostic assay may also be used todetermine the ratio of expression between ASIC2A and ASIC3, and anychanges in these expression rations, as an index or marker of ASIC-2S.2expression.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences encodingASIC2A or ASIC3 or closely related molecules, may be used to identifynucleic acid sequences which encode ASIC2A or ASIC3. The specificity ofthe probe, whether it is made from a highly specific region, e.g., 10unique nucleotides in the 5′ regulatory region, or a less specificregion, e.g., especially in the 3′ coding region, and the stringency ofthe hybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding ASIC2A or ASIC3, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe ASIC2A or ASIC3 encoding sequences. The hybridization probes of thesubject invention may be DNA or RNA and derived from the nucleotidesequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO:7, orfrom genomic sequences including promoter, enhancer elements, andintrons of the naturally occurring ASIC2A and ASIC3.

Means for producing specific hybridization probes for DNAs encodingASIC2A or ASIC3 include the cloning of nucleic acid sequences encodingASIC2A or ASIC3 derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, commercially available, andmay be used to synthesize RNA probes in vitro by means of the additionof the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, radionuclides such as ³²P or ³⁵S, orenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

Polynucleotide sequences encoding ASIC2A and/or ASIC3 may be used forthe diagnosis of conditions or diseases which are associated withexpression of ASIC-2S.2. Examples of such conditions or diseases includeneurological diseases including chronic pain, neuropathic pain such asdiabetic-, cancer-, and AIDS-related neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, Huntington's disease,Creutzfeld-Jacob disease, and amyotrophic lateral sclerosis, anddementias, such as AIDS-related, as well as convulsions, epilepsy,stroke, and anxiety and depression, cardiovascular diseases such asangina, congestive heart failure, vasoconstriction, hypertension,atherosclerosis, restenosis, and bleeding. The polynucleotide sequencesencoding ASIC2A or ASIC3 may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; orin dip stick, pin, ELISA or chip assays utilizing fluids or tissues frompatient biopsies to detect altered ASIC-2S.2 expression. Suchqualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding ASIC2A and/orASIC3 may be useful in assays as probes that detect activation orinduction of various neurological or other non-neurological disorders,particularly those mentioned above. The nucleotide sequence encodingASIC2A and/or ASIC3 may be labelled by standard methods and added to afluid or tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantified and comparedwith a standard value. If the amount of signal in the biopsied orextracted sample is significantly different from that of a comparablecontrol sample, this indicates that the levels of nucleotide sequencesthat hybridized with the labelled probe in the sample are alsodifferent. The presence of altered levels of nucleotide sequencesencoding ASIC2A and/or ASIC3 in the sample indicates the presence of theassociated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of ASIC-2S.2, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes ASIC2A and/or ASIC3 underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to neurological diseases, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventive measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the disease.

Additional diagnostic uses for oligonucleotides encoding ASIC2A and/orASIC3 may involve the use of PCR. Such oligomers may be chemicallysynthesized, generated enzymatically, or produced from a recombinantsource. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation and another with antisense, employed underoptimised conditions for identification of a specific gene or condition.The same two oligomers, nested sets of oligomers, or even a degeneratepool of oligomers may be employed under less stringent conditions fordetection and/or quantification of closely related DNA or RNA sequences.

Methods which may also be used to quantify the expression of ASIC-2S.2include radiolabelling or biotinylating nucleotides, coamplification ofa control nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby P C et al. J Immunol Methods, 1993; 159:235; Duplaa C et al. Anal Biochem 1993; 229). The speed ofquantification of multiple samples may be accelerated by running theassay in an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or calorimetric responsegives rapid quantification.

Screening Assays

In another embodiment of the invention, ASIC-2S.2, its active, catalyticor immunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenASIC-2S.2 and the agent being tested, may be measured. Thus, thepolypeptides derived from ASIC-2S.2, or any component thereof, may alsobe used to assess the binding of small molecule substrates and ligandsin, for example, cells, cell-free preparations, chemical libraries, andnatural product mixtures. These substrates and ligands may be naturalsubstrates and ligands or may be structural or functional mimetics. Ingeneral, such screening procedures involve producing appropriate cells,which express the receptor polypeptide complex of the present inventionon the surface thereof. Such cells include cells from mammals, yeast,insects (e.g. Drosophila) or bacteria (e.g. E. coli). Cells expressingthe receptor (or cell membranes containing the expressed receptor) arethen contacted with a test compound to observe binding, or stimulationor inhibition of a functional response (for example inhibition ofproton-activated currents).

The assays may simply test binding of a candidate compound whereinadherence to the cells bearing the receptor is detected by means of alabel directly or indirectly associated with the candidate compound orin an assay involving competition with a labelled competitor. Further,these assays may test whether the candidate compound results in a signalgenerated by activation of the receptor, using detection systemsappropriate to the cells bearing the receptor at their surfaces (forexample increased ion permeation measured by patch clamp or, preferablyby ion imaging with ion-specific dyes). Inhibitors of activation aregenerally assayed in the presence of a known agonist (for exampleprotons) and the effect of the candidate compound on the activation bythe agonist is observed. Standard methods for conducting such screeningassays are well understood in the art. Typically, the response may bemeasured by use of a microelectrode technique accompanied by suchmeasurement strategies as voltage clamping of the cell wherebyactivation of ion channels may be identified by inward or outwardcurrent flow as detected using the microelectrodes. ²²Na, ⁸⁶Rb, ⁴⁵Caradiolabelled cations or ¹⁴C or ³H guanidine may be used to assess suchion flux; a sodium, calcium or potassium ion sensitive dye (such asFura-2, or Indo) may also be used to monitor ion passage through thereceptor ion channel, or a potential sensitive dye may be used tomonitor potential changes, such as in depolarisation.

Alternatively, it is also possible to mutate the ASIC2A and/or ASIC3cDNA in order to produce a constituvely active ASIC-2S.2 channel, as hasbeen shown with other DEG/ENaC family members (Huang et al., Nature 367:467; Waldman et al., J Biol Chem 1997: 271; 10433, Sakai et al., JPhysiol 1999; 519: 323, Schaefer et al., FEBS Lett 2000; In Press).Then, the constitutively active channel may be expressed in host cellsto produce a screening assay where channel activity is permanent. Therecording of channel activity my be carried out either by membranevoltage analysis, directly (patch clamp, for example) or indirectly(fluorescent probes, for example) or by sodium entry measurement(radioactive sodium influx, fluorescent probes, or reporter genes).

Another technique for drug screening, which may be used, provides forhigh throughput screening of compounds having suitable binding affinityto the protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to ASIC-2S.2, large numbers ofdifferent small test compounds are synthesised on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with ASIC-2S.2, or components, or fragments thereof, and washed.Bound ASIC-2S.2 is then detected by methods well known in the art.Purified ASIC-2S.2, or any component thereof, can also be coateddirectly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralising antibodies can be used tocapture the peptide and immobilise it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralising antibodies capable of binding ASIC-2S.2 specificallycompete with a test compound for binding ASIC-2S.2. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with ASIC-2S.2.

In additional embodiments, the nucleotide sequences which encode theindividual components of ASIC-2S.2, namely ASIC2A and ASIC3, may be usedin any molecular biology or pharmacology techniques that have yet to bedeveloped, provided the new techniques rely on properties of thenucleotide or polypeptide sequences that are currently known, including,but not limited to, such properties as the triplet genetic code andspecific base pair interactions.

EXAMPLES

The following examples are intended to further illustrate the inventionand are not intended to limit the scope of the invention in any way. Allreferences cited herein, whether previously or in the followingexamples, are expressly incorporated in their entirety by reference. Alloligonucleotides disclosed in the following examples are designed usingtwo recognised software packages: GeneWorks 2.5.1 and MacVector 6.0.1(Oxford Molecular).

Example 1 Vector Constructs for the Functional Expression of ASIC-2S.2Channels

Method 1: Expression of ASIC-2S.2 is accomplished by introducing intoappropriate host cells, by various injection or transfection techniquesknown to one skilled in the art, two separate vectors comprising thenucleic acids encoding, respectively, the ASIC2A and ASIC3 polypeptidesof SEQ ID NO:2 or NO:6 and SEQ ID NO: 4 or NO:8. A preferred eukaryoticexpression vector is pcDNA3 (InVitrogen), or any derivatives thereof.Based on nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5and SEQ ID NO:7, specific oligonucleotide primers are designedimmediately upstream and downstream, respectively, of the initiation andthe stop codons. All primers are extended to add artificial restrictionsites (e.g. forward primers with EcoRI and reverse primers with XbaI),allowing RT-PCR amplified full length nucleic acids to be directionallysubcloned into pcDNA3. Ligated products are used to transform E. Colistrain DH5α from which purified plasmids are prepared using commerciallyavailable kits (Quiagen).

Method 2: Expression of ASIC-2S.2 is achieved by introducing intoappropriate host cells, by various injection or transfection techniquesknown to one skilled in the art, a biscistronic vector comprising twonucleic acids encoding, respectively, ASIC2A and ASIC3 polypeptides ofSEQ ID NO:2 or NO:6 and SEQ ID NO: 4 or NO:8. Bicistronic expressionvectors produce one transcript with two translation initiation points,resulting in the simultaneous expression of two genes of interest. Apreferred bicistronic vector is pIRES (Clontech) which permits thesubcloning of two distinct genes in two separate multiple cloning sites(MCS A and B) located on either side of the internal ribosome entry site(IRES) from the encephalomyocarditis virus (ECMV). This allows thetranslation of two consecutive open reading frames from the samemessenger RNA (Jang S K et al. J. Virol. 1990; 62: 2636—Rees S et al.:BioTechniques 1996; 20: 102). The MCSs and IRES sequences are downstreamof the immediate early promoter of cytomegalovirus (PCMV IE). Theintervening sequence (IVS) between PCMV IE and the MCS is an intron thatis efficiently spliced out following transcription. SV40 polyadenylationsignals downstream of the MCS direct proper processing of the 3′ end ofthe mRNA from subcloned genes. Bacteriophage T7 and T3 promoters arelocated upstream and downstream of MCS A and B, respectively. pIRES usesthe neomycin resistance gene (Neor) to permit selection of transformedcells. Neor is expressed from the SV40 enhancer/promoter, and asynthetic polyadenylation signal directs proper processing of the 3′ endof the Neor mRNA. The SV40 origin also allows for replication inmammalian cells expressing the SV40 T antigen. The vector backbone alsocontains the β-lactamase gene for ampicillin resistance and a ColE1origin of replication for propagation in E. coli and a f1 origin forsingle-stranded DNA production. As described above in method 1, RT-PCRamplified ASIC2A and ASIC3 nucleic acids are directionally subcloned,respectively, into MCS A and MCS B (or vice versa). Control vectorscomprising two copies of ASIC2A- or ASIC3-encoding nucleic acids arealso constructed. Ligated products are used to transform E. Coli strainDH5α from which purified plasmids are prepared using commerciallyavailable kits (Quiagen).

Method 3: Expression of ASIC-2S.2 is achieved by introducing intoappropriate host cells, by various injection or transfection techniquesknown to one skilled in the art, an expression vector comprising anengineered chimeric nucleic acid which encodes both ASIC2A and ASIC3polypeptides as a single tandem polypeptide delimited by the initiationmethionine of the first subunit and the stop of the second subunit. Thefollowing example illustrates this method: The full length codingsequence of human ASIC3 is subcloned into pCDN3 between HindIII andEcoRI sites (plasmid A), while ASIC2A coding nucleic acid sequence issubcloned between EcoRI and XbaI (plasmid B). An ASIC3-specific forwardprimer just upstream of a natural NcoI site (SEQ ID NO:9) is paired witha mutagenic oligonucleotide primer designed to eliminate the stop codonand add an artificial EcoRI site (SEQ ID NO:10). A 570 bp fragment isamplified by PCR using the proof-reading DNA polymerase pfu(Stratagene). Following digestion with NcoI and EcoRI, the purifiedfragment is then back-cloned into the ASIC3-containing plasmid A inreplacement of the corresponding wild type fragment. In the second step,the above mutated ASIC3 full length nucleic acid is cut out from thevector with HindIII and EcoRI and subcloned immediately upstream and inframe of the ASIC2A coding sequence of plasmid B. A number of differentstrategies can be designed to prepare similar constructs and theprevious example is intended solely to illustrate this method and not tolimit its scope in any way. The method also encompasses constructs whereASIC2A is placed upstream of ASIC3 as well as constructs where more thantwo subunits are attached together in any pertinent combination andsynthesised as a single polypeptide.

Example 2 Expression of Functional ASIC-2S.2 Channels in Xenopus laevisOocytes

According to method 1 described above, nuclei of Xenopus oocytes areinjected with ASIC2A and ASIC3 cDNAs separately subcloned into pcDNA3expression vector (1-5 ng). Control oocytes are injected with H₂O.Oocytes are maintained at 18° C. in modified Barth's solution.Proton-activated currents are measured by two-electrode voltage clamp1-3 days after injection. During voltage clamp (−60 mV/−100 mV)),oocytes are bathed in 116 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 10 mM aceticacid and 5 mM Hepes (pH 7.4 with NaOH). To determine proton-gating, bathsolution is quickly switched to a solution of pH<7.4 for 10 sec, thenreturned to bath solution for washout. The stimulating solution isprepared by lowering the pH of the original bath solution withhydrochloric acid. The osmolality of the solutions is verified with anosmometer and corrected with mannitol or choline chloride. To documentionic selectivity, NaCl is replaced with LiCl or KCl. Current-voltagerelationships are determined by stepping from a holding potential of −60mV to potentials between −100 and +60 mV for 10 seconds before andduring stimulation with low pH solution. FIGS. 1A (human) and 1B (rat)compare proton-activated currents carried by homomultimeric ASIC2A andASIC3 receptors to the heteromultimeric ASIC-2S.2 receptors (ASIC2a+3).The much greater amplitude and the consistent biphasic profile of thecurrent recorded in co-injected oocytes clearly reveals the existence ofa novel proton-gated channel resulting from the assembly of ASIC2A andASIC3. Species differences are also noteworthy. Indeed, using humansubunits, both the early fast and sustained currents are stronglypotentiated, while in the case of rat, the major effect appears to be onthe sustained current only.

FIG. 2 compares pH-response curves of homomultimeric andheteromultimeric ASIC channels of the present invention.

The oocyte expression system is also used to test and screen forcompounds with agonist or antagonist activity. FIGS. 5A and 5Billustrate this principal by showing the inhibitory effects of amilorideand gadolinium on proton-activated currents.

Example 3 Tissue Distribution of ASIC2A and ASIC3 Transcripts UsingRT-PCR

To document the co-expression of ASIC2A and ASIC3 mRNA in varioustissues, specific oligonucleotide primers are designed and used in aduplex RT-PCR protocol. Fragments of 470 and 340 bp are amplified,respectively, with ASIC2A-specific (SEQ ID NO: 11 and SEQ ID NO: 12) andASIC3-specific (SEQ ID NO:13 and SEQ ID NO:14) primers, enabling theco-amplification of both fragments from a single sample. The reaction iscarried out with the EXPAND long-template polymerase mix, containingboth Taq and Pwo polymerases (Roche), according to the manufacturer'sinstructions. Briefly, the reaction mix includes: dNTPs 0.5 mM, forwardand reverse primers 1 μM each, RT-cDNA template 5 μL, 10×PCR buffer 5 μLand polymerase enzyme mix 0.75 μL, all in a final volume of 50 μL.Samples are kept at 4° C. and the enzyme mix is added last. Tubes arethen immediately transferred to the thermocycler preheated to 94° C.,after which cycling is launched. Typical cycling conditions are asfollows: Initial denaturation step: 2 min at 94° C., than 40 cycles of45 sec at 94° C., 45 sec at 58° C. and 2 min at 72° C., followed by afinal extension step of 10 min at 72° C. RT-cDNAs are either commercial(Clontech) or prepared from RNA or mRNA either with Superscript orThermoscript enzyme mixes, according to the manufacturer's directions(Gibco Life Sciences). RNA and mRNA are prepared using standardmolecular biology protocols, such as decribed in Maniatis et al., (seeabove) or using commercially available kits, such as the S.N.A.P. totalRNA isolation kit, Fast Track 2.0 and micro Fast Track 2.0 mRNAisolation kits (InVitrogen). An example of tissue distribution ofhASIC2A and hASIC3 mRNA expression appears in FIG. 4. Trigeminal gangliaare among tissues with the highest coexpression, suggesting that theheteromultimeric ASIC-2S.2 channel might be involved in pain and/orsensory transmission. This constrasts with previous results reported forrat where ASIC2A is apparently not expressed in sensory neurons.

Example 4 Co-localisation of ASIC2A and ASIC3 Transcripts Demonstratedby In Situ Hybridization

Hybridization probes derived from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5and/or SEQ ID NO:7 are employed to screen cDNAs, genomic DNAs, or mRNAs.An example of such use is in situ hybridization to mRNAs. Briefly, a 278bp fragment corresponding to nucleotides 181-459 of rat ASIC2A(Ser⁶¹-Met¹⁵³) is subcloned between Sac I and Sph I sites of the pGEM5zfvector. A 378 bp fragment of rat ASIC3, corresponding to nucleotides1142-1520 of ASIC3 (Leu³⁸¹-Pro⁵⁰⁷), is subcloned between Sac I and Apa Isites of the pGEM5zf vector. Sense and antisense cRNAs are synthesizedwith the SP6 and T7 RNA polymerases in the presence of [³²P]-UTP forNorthern blot or a mix of [³⁵S]-CTP and [³⁵S]-UTP for in situhybridization. For the latter, 6 ηm-thick tissue sections are fixed for1 hr with 4% formaldehyde in 0.1 M phosphate buffer (pH 7.2) and washedextensively with phosphate buffered saline (PBS), then reacted withacetic anhydride in triethanolamine 0.1 M solution. Then, the sectionsare hybridized overnight at 55° C. using the double [³⁵S]-CTP and[³⁵S]-UTP-labelled cRNA probes. After extensive washing, the sectionsare dried and exposed to X-ray film for 2-3 days (Marcinkiewicz et al.Neuroscience 1997; 76: 425). FIG. 5 shows an example in rat cerebellumwhere ASIC2A and ASIC3 positive grains are clearly present on the samecell type, namely the Golgi cells (GC) of the granular cell layer.

Example 5 Co-purification of ASIC2A and ASIC3 Subunits

The existence of a novel heteromultimeric proton-gated ion channel,initially revealed by electrophysiological data, is further corroboratedby biochemical data providing direct evidence of the association betweenASIC2A and ASIC3 subunits through protein/protein interactions. For thispurpose, N- or C-terminal epitope-tagged fusion proteins are constructedwith ASIC2A and ASIC3. For the C-terminus, mutagenic oligonucleotideprimers eliminate the stop codons of ASIC2A and ASIC3 and respectivelyadd an artificial XhoI and SaII restriction site to the 3′ end. Then,the PCR-amplified full-length ASIC2A and ASIC3 cDNAs are subclonedbetween the artificial EcoRI-XhoI sites into a pcDNA3 vector containingan in frame cassette with a FLAG or His₆ epitope followed by anartificial stop codon. N-terminal His₆ tagging of ASIC3 is achieved bydirectly subcloning a full-length EcoRI-NotI ASIC3 fragment into thepcDNA3.1/HisA vector (Invitrogen). Similarly, a PCR-amplifiedfull-length ASIC2A cDNA flanked with EcoRI-XhoI sites is subcloned intothe pcDNA3.1/HisB vector (InVitrogen) as well as in the pEGFP-C1 vector(Clontech), providing, respectively, a N-terminal His₆- and GFP-taggedASC2A. All the above ASIC2A and ASIC3 derivatives were done with humansubunits, but a similar approach can also be done for the rat subunits,or any other species, by anyone skilled in the art. All new taggedreceptors are tested for function before performing co-purificationexperiments. The results from the functional tests are summarised inFIG. 6B. For co-purification, the following combinations are eitherco-injected into oocytes or co-transfected into HEK 293 cells fortransient expression:

-   -   1) ASIC2A-N_His₆+ASIC3-C_FLAG    -   2) 2) ASIC2A-N_GFP+ASIC2A-N_His₆    -   3) ASIC2A-N_GFP+ASIC3-N_His₆.    -   (Where “N_” indicates N-terminal tagging and “C_” indicates        C-terminal tagging).

After the required time for protein expression, oocytes or cells arecollected, lysed and membranes solubilized in a Triton X-100-containingbuffer (Tinker et al., Cell 1996; 87: 857—Lê et al., J Neuroscience1998; 18: 7152). Unsolubilized material is removed by centrifugation andsupernatants are incubated with a Ni-NTA-resin (Quiagen) (2 and 3) or animmunoaffinity resin coupled with antiFLAG M2 monoclonal antibodies.After several washes, bound proteins are either eluted with 500 mMimidazole (Ni-NTA) or directly with the SDS-PAGE loading buffer.Proteins separated by gel electrophoresis are transferred ontonitrocellulose membranes (Amersham) and immunoprobed with commerciallyavailable antibodies against GFP (Green Fluorescent Protein) or His₆epitopes, followed by peroxidase-labelled secondary antibodies forchemiluminescence detection (ECL kit, Amersham). Results from theseexperiments appear in FIGS. 7A (HEK 293 cells) and 7B (oocytes) andprovide for the first time ever the proof of direct protein-proteininteractions between ASIC2A and ASIC3 polypeptides. Indeed, bothsubunits are always purified together, independently of which subunit isinitially targeted by the purification step. We have therefore provideddirect biochemical evidence for the existence of the novelheteromultimeric ASIC-2S.2 receptor.

Example 6 Screening for Compounds Capable of Modulating Ion ChannelActivity and/or Properties

As described in example 2, voltage clamped oocytes expressing theASIC-2S.2 channels are used to screen for molecules capable ofmodulating, activating or inhibiting channel activity. See FIGS. 3A and3B.

Alternatively, permanently or transiently transfected or infected celllines (e.g. COS, HEK 293) expressing ASIC-2S.2, cultured in multiwellplates, are loaded with potential- or cation-sensitive (sodium, calcium)dyes and the fluorescence emission is measured following application ofcontrol and low pH buffers (e.g. pH 7.4 and pH 5.0). The responses toboth buffers in the presence and absence of candidate compounds arecompared to identify compounds, which stimulate, inhibit or modulateASIC-2S.2.

Particular subclasses of channel antagonists are substances capable ofdisrupting protein-protein interactions between the subunits, whichcompose the ASIC-2S.2 channels. These compounds will either prevent theassociation of ASIC2A and ASIC3 into functional channels or dissociatealready assembled ASIC-2S.2 receptors by binding to the specific proteindomains responsible for ASIC subunit interactions. We have evidenceindicating that the intracellular domains of ASIC2A and/or ASIC3 areinvolved in the assembly of functional channels. The C-termini appear tobe particularly important since modifications to this domain generatesnon-functional subunits in both homo- and heteromultimeric form. Indeed,as seen in FIG. 6B, all N-terminally tagged ASIC2A or ASIC3 retain fullfunction and ability to interact with a functional counterpart. Incontrast, C-terminal tagging in most cases created non-functional,non-interacting subunits. Therefore, fusion proteins are constructedcomprising only the intracellular domains of human ASIC2A or humanASIC3. Briefly, C-terminal fragments of ASIC3 and ASIC2A are amplifiedwith mutagenic primers (SEQ ID NO:15 and NO:16 for hASIC3; SEQ ID NO:17and NO:18, for hASIC2A) that add an in frame artificial initiation siteat the 5′ end, as well as artificial HindIII and BamHI restrictionsites, used for subcloning of said fragments into pcDNA3 expressionvectors. The resulting C-terminal nucleic acid and peptide fragments areindicated respectively in SEQ ID NO: 19 and SEQ ID NO: 20, for humanASIC3 and SEQ ID NO 21 and SEQ ID NO: 22 for human ASIC2A. Additionally,N-terminal fragments of ASIC3 and ASIC2A are constructed, by insertingan artificial stop codon with mutagenic primers (SEQ ID NO:23 and itsreverse for hASIC3; SEQ ID NO:24 and its reverse for hASIC2A) using thecommercially available mutagenesis kit QuickChange (Stratagene™),according to the manufacturer's directions. All fragments are insertedinto the pcDNA expression vector. The resulting N-terminal nucleic acidand peptide fragments are indicated respectively in SEQ ID NO: 25 andSEQ ID NO: 26 for human ASIC3 and SEQ ID NO: 27 and SEQ ID NO: 28 forhuman ASIC2A. The inhibitory effects of these fragments on channelfunction and/or assembly is tested by co-expressing these truncatedconstructs together with wild-type ASIC2A and/or ASIC3 both in homomericand heteromeric combinations (see FIGS. 9A and 9B). Additional fusionproteins with shorter fragments of the intracellular domains are used toidentify the smallest amino acid sequence involved in the interaction ofASIC2A and ASIC3. This sequence of amino acids is validated by theinhibitory and/or disruptive effect of small peptides corresponding tothe identified amino acid sequence, when said peptides are introducedinto hosts expressing the ASIC-2S.2 heteromultimeric channel. A drugscreening method based on the identified peptides is used to identifymolecules capable of binding to them. Such compounds will, in turn, bindto the corresponding amino acid sequence present in the full-lengthwild-type subunits and inhibit therefore subunit assembly. A number ofapproaches can be used for this purpose. For example, candidatecompounds previously arrayed and attached to multi-well plates areexposed to the above-described peptides, linked to an additionalepitope. After washing steps, wells holding compounds that bind thespecific amino acid sequence are revealed with an ELISA-type based assayusing labelled antibodies against the grafted epitope Data obtainedusing different concentrations of the peptides are used to calculatevalues for the number, affinity, and association constants of theinteraction.

A similar approach as described above is also used with the N-terminalfragments of ASIC2A and/or ASIC3. Briefly, mutagenic primers aredesigned to introduce an artificial stop codon just upstream of thesequence encoding the first putative transmembrane domain of ASIC2A orASIC3. Using commercially available mutagenesis Kits (e.g. QuickChange,Stratagene), the above mutations are incorporated into the plamidscomprising the ASIC2A or ASIC3 nucleic acids. When these mutatedplasmids are introduced into relevant hosts, only the first portioncorresponding to the N-terminal intracellular domain of the ASIC2Aand/or ASIC3 polypeptides is translated. Coexpression of said fragmentswith full length ASIC2A and ASIC3 is done to document their effect onthe assembly of ASIC-2S.2 channels. Drug screening methods based on thesmallest active N-terminal fragment are performed as described above forthe C-terminal fragments.

Example 7 Antisense Molecules

Antisense molecules to the sequences encoding the ASIC-2S.2 components,or any part thereof, are used to inhibit in vivo or in vitro expressionof naturally occurring ASIC-2S.2. Although use of antisenseoligonucleotides, comprising about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. An oligonucleotide based on the coding sequences of eitherASIC2A or ASIC3 are used to inhibit expression of naturally occurringASIC-2S.2. The complementary oligonucleotide is designed from the mostunique 5′ sequence of the coding region and used either to inhibittranscription by preventing binding to the upstream untranscribedsequence or translation of an ASIC2A- or ASIC3-encoding transcript bypreventing ribosomes from binding. Using an appropriate portion of the5′ sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7,an effective antisense oligonucleotide includes any 15-20 nucleotidesspanning the region which translates into the 5′ coding sequence of thepolypeptides of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO:8.

All publications mentioned in the above specifications are hereinincorporated by reference. Various modifications and variations of thedescribed method and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology, electrophysiology or related fields are intended tobe within the scope of the following claims.

1. A purified heteromultimeric acid-sensing ion channel comprising: (a)an ASIC2A polypeptide comprising an amino acid sequence at least 90%identical to the amino acid sequence set forth in SEQ ID NO:2 or SEQ IDNO:6, and (b) an ASIC3 polypeptide comprising an amino acid sequence atleast 90% identical to the amino acid sequence set forth in SEQ ID NO:4or SEQ ID NO:8, wherein the cation permeability of said channel isinduced by an increase in proton ion concentration.
 2. The purifiedheteromultimeric acid-sensing ion channel of claim 1, wherein the aminoacid sequence of the ASIC2A polypeptide is at least 95% identical to theamino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:6.
 3. Thepurified heteromultimeric acid-sensing ion channel of claim 1, whereinthe amino acid sequence of the ASIC2A polypeptide is at least 97%identical to the amino acid sequence set forth in SEQ ID NO:2 or SEQ IDNO:6.
 4. The purified heteromultimeric acid-sensing ion channel of claim1, wherein the amino acid sequence of the ASIC3 polypeptide is at least95% identical to the amino acid sequence set forth in SEQ ID NO:4 or SEQID NO:8.
 5. The purified heteromultimeric acid-sensing ion channel ofclaim 1, wherein the amino acid sequence of the ASIC3 polypeptide is atleast 97% identical to the amino acid sequence set forth in SEQ ID NO:4or SEQ ID NO:8.
 6. The purified heteromultimeric acid-sensing ionchannel of claim 2, wherein the amino acid sequence of the ASIC3polypeptide is at least 95% identical to the amino acid sequence setforth in SEQ ID NO:4 or SEQ ID NO:8.
 7. The purified heteromultimericacid-sensing ion channel of claim 2, wherein the amino acid sequence ofthe ASIC3 polypeptide is at least 97% identical to the amino acidsequence set forth in SEQ ID NO:4 or SEQ ID NO:8.
 8. Theheteromultimeric acid-sensing ion channel of claim 1, wherein the ASIC2Apolypeptide comprises the amino acid sequence SEQ ID NO:2 or SEQ ID NO:6and the ASIC3 polypeptide comprises the amino acid sequence SEQ ID NO:4or SEQ ID NO:8.
 9. A process for producing a heteromultimericacid-sensing ion channel comprising, culturing a host cell thatexpresses a recombinant ASIC2A polypeptide comprising an amino acidsequence at least 90% identical to the amino acid sequence set forth inSEQ ID NO:2 or SEQ ID NO:6, and a recombinant ASIC3 polypeptidecomprising an amino acid sequence at least 90% identical to the aminoacid sequence set forth in SEQ ID NO:4 or SEQ ID NO:8, under conditionssufficient to express the recombinant ASIC2A and ASIC3 polypeptides. 10.A method of identifying an agonist of the heteromultimeric acid-sensingion channel of claim 1, comprising: (a) contacting a cell expressing theheteromultimeric acid-sensing ion channel of claim 1 with a candidatecompound; and (b) determining the ion permeability of theheteromultimeric acid-sensing ion channel in the presence and absence ofthe candidate compound, wherein an increase in the ion permeability inthe presence of the candidate compound indicates that the candidatecompound is an agonist of the heteromultimeric acid-sensing ion channel.11. A method of identifying an antagonist of the heteromultimericacid-sensing ion channel of claim 1, comprising: (a) contacting a cellexpressing the heteromultimeric acid-sensing ion channel of claim 1 witha candidate compound in the presence of proton ions; and (b) determiningthe ion permeability of the heteromultimeric acid-sensing ion channel inthe presence and absence of the candidate compound, wherein an decreasein the ion permeability in the presence of the candidate compoundindicates that the candidate compound is an antagonist of theheteromultimeric acid-sensing ion channel.